Revision as of 23:18, 26 October 2012

PCR mutation

In reference to the PCR Mutation protocol, 100 ng/μL of the primer was needed for the reaction. The weight of the primer in the provided container was 0.46 mg. A ratio of the weight over volume was equated to the required concentration of the primer:

0.46 mg = 0.46E6 ng

= of primer in water = 4600 μL of water

There is limited space in the plastic container (1 mL). Instead of dissolving 0.46E6 ng of the primer in 4600 μL of water, the entire primer was dissolved in 1 mL water.

Using M1V1 = M2V2, the volume taken from the solution of 0.46E6 ng in 1 mL of water was calculated to be 217.39 μL. This was transferred to a new tube and filled up with water to a total volume of 1 mL.

V1 = = 217.39 μL of the dissolved primer in water

The procedure listed in PCR Mutation protocol was strictly followed. After the addition of all reagents, the sample was placed in the thermocycler.

Continuation of Chemiluminescence

The luminol prepared from the previous chemiluminescence laboratory period had a pH of 7 to 8. Reviewing a journal article of Xiaoyu, it was determined that the optimal pH of luminol was 12.5. As a result, it was decided to prepare a new solution of luminol at a pH of 10 to 11.

A weight of .0112 g of luminol was added to 6 mL of water. The buffer composed of a direct addition of .0737 g of sodium carbonate and .4358 g of sodium bicarbonate.

Using a pH meter, the electrode detected the pH at 8.73. As suggested by Dr. Hartings, a solution of sodium carbonate was made to increase the pH of the solution.

Several adjustments were made in increasing the pH. A total weight of 1.91 g of sodium carbonate dissolved in 15 mL of water was added to the 6 mL solution of luminol. The final pH for luminol was 10.55.

The concentration of luminol has been diluted by the addition of 15 mL of water.

Molarity of diluted luminol = = 2.85714 mM of luminol

Since there was limited amounts of the solid form of luminol, it was decided to take 6 mL of the 2.86 mM luminol stock solution and then add the appropriate amount of luminol to the 6 mL volume. The molarity of the 6 mL solution was very minute; the molarity was approximated as 2 mM. By making this assumption, .00106 g of luminol would make a 1 mM solution in 6 mL of water; this amount was multiplied by 8. The product was 0.00848 g of luminol was needed to be added into the 2 mM solution of luminol to increase the molarity to 10 mM. The amount was weighed and added to the 6 mL 2.86 mM solution of luminol.

Due to time constraints, the chemiluminescence of the luminol at pH 10.55 was tested on a lab bench with the room lights turned off. The reaction produced a neon blue glow that lasted for more than 3 minutes. No photograph was taken since the apparatus of the camera was not suited for the fluorescence activity.